| Objective: Gliomas, especially high-grade gliomas are the most common malignant brain tumor showed infiltrative growth. It's very difficult to cut the tumor completely. The overall prognosis of glioma patients is still not ideal in spite of comprehensive treatment including radiotherapy and chemotherapy. The recurrence rate of glioma was high and the survival time for patients with high-grade glioma were even less than 1 year. With the continuous development of molecular biology, glioma immunotherapy is arousing people's attention gradually. The aggressive nature of this neoplasia is closely related to its complex pathophysiology. In particular, evasion of the immune system by gliomas limits effective anti-tumoral responses. Current evidence suggests a major role in the evasion of immune rejection by CD4~+CD25~+ regulatory T cells (Tregs).Tregs are lymphocytes that have a physiological role in the modulation of the immune response. Specifically, these cells prevent autoimmunity by inhibiting autoreactive effector T lymphocytes, which downregulates the anti-tumoral response and promotes the growth of tumor. Many studies have found a large population of Tregs were infiltrated in the tissue and blood of many malignancies including breast, ovarian, lung, liver carcinoma and malignant lymphoma. It's suggested that the population of Tregs was correlated negatively with the progression and prognosis of tumor. Depletion of Tregs or inhibition of its function has been associated with recovery of anti-tumoral response.Tregs are divided into thymus-derived Tregs or natural Tregs (nTregs) and Inducible Tregs (iTregs). nTreg development takes place in the thymus and iTregs are very similar in function to nTregs but derive from non-Treg cells in the periphery under specific stimulation. The abnormal increasing Tregs in tumor microenvironment are also divided into two groups. One group includes Tregs migrated from periphery under the influence of chemokines. The other group are induced from CD4~+CD25–T cells upon the exposure to the suppressive cytokine milieu at the tumor site. Tregs separated from tumor infiltrated lymphocytes usually represent tumor-specific antigen. The differentiation and maturation of Tregs take place under the action of tumor cells, which prevent the function of effector T cells (mainly CD8~+, CD4~+). Imbalance between Tregs and conventional T cells play a role in the immune escape of tumor cells and recovery of imbalance may be a new strategy for tumor treatment.As pro-inflammatory cytokine, interleukin 18(IL-18) has also been implicated in multiple autoimmune associated disease, such as rheumatoid arthritis, sarcoidosis, lupus erythematosus. Tregs have been reported to be decreased or to have decreased functional activity in a number of autoimmune disorders, and it is possible that an imbalance of Tregs and effector T cells due to differential IL-18 signaling contributes to the loss of tolerance. Several preclinical studies have suggested that IL-18 may have significant antitumor effects. IL-18 enhances the production of IFN-γby T cells and NK cells and can augment the cytolytic activity of NK cells and cytotoxic T lymphocytes (CTL). IL-18 promotes the differentiation of activated CD4 T cells into helper effector cells of Th1 or Th2 type. It's not clear, however, that IL-18 can manipulate the function of Tregs in tumor microenvironment.Aim of our investigation is to discuss the expression of Tregs in cancer tissue from patients with glioma, the correlation between the population of Tregs and progression of tumor, investigate the inhibitory mechanism of Tregs for immune fanction, analyze the manipulation of IL-18 on Tregs.Methods:1Tumor tissue and blood of 76 patients with astrocytoma were collected, who were operated in the department of neurosurgery of the 4th hospital affiliated to Hebei Medical University from September, 2007 to September, 2009. Normal brain tissue and blood were used for control, which were from patients with decompression surgery for herniation and health volunteers. TILs and PBMCs were separated from tumor tissue and blood. The frequency of Tregs, CD4~+T cells and CD8~+T cells and ratio between Tregs and other T cells in TILs and PBMC were detected by flow cytometry. Then, the frequency and ratio of Tregs were analyzed and observe the relationship between Tregs and clinical pathological characters, prognosis of patients with astrocytoma.Expression of TGF-β1 in periphery blood were analyzed by ELISA. The Foxp3mRNA in PBMC were detected by RT-PCR, and analyze the relationship between the expression of Foxp3mRNA and TGF-β1.2 Tumor tissue and blood of 5 patients with glioblastoma were collected, who were operated in the department of neurosurgery of the 4th hospital affiliated to Hebei Medical University from September, 2008 to September, 2009. Normal brain tissue and blood were used for control, which were from patients with decompression surgery for herniation and health volunteers. Primary glioma cells and primary normal astrocyte were cultured in vitro and identified by immunohistochemistry, respectively. Primary glioma cells and primary normal astrocytes were cultured successfully in vitro, and identified by immunohistochemistry. CD4~+CD25~+T cells, CD4~+CD25-T cells and CD8~+T cells from TIL and PBMC were isolated by immunomagnetic separation system. After magnetic activated cell sorting, the purity of CD4~+CD25-, CD8~+T cells was 89~92%, higher than 90% and 87~94%, respectively.CD4~+CD25-T cells were then co-cultured with CD4~+CD25~+T cells in different population. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells.Expression of Foxp3, CTLA-4, GITR, CCR4 and CCR8 protein were detected by West Blot. The secretion of TGF-β1, CCL22 and CCL12 in the medium supernatant of primary glioma cells, primary normal astrocyte and different CD4~+CD25~+T cells.3 30 male Fisher344 rats were divided randomly into 2 groups: 9L group and IL-18/9L group.9L cells and IL-18/9L cells were inoculated into the right cerebral hemisphere, respectively. Three mice of each group were killed on the 7th, 14th and 21st day after inoculation of tumor cells, the spleens and tumors were obtained from mice. 6 rats remained of every group were observed for over survival time.Expression of IL-18mRNA in rat tumor tissue were detected by RT-PCR. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells. The frequency of Tregs, CD4~+T cells and CD8~+T cells and ratio between Tregs and other T cells in TILs and PBMC were detected by flow cytometry on 21st day after inoculation of tumor cells. IFN-γproducted by splenocytes were detected with ELISA.On 21st day after inoculation of tumor cells, CD4~+CD25~+T cells, CD4~+CD25-T cells and CD8~+T cells from TIL and PBMC of rats were isolated by immunomagnetic separation system. CD4~+CD25-T cells were then co-cultured with CD4~+CD25~+T cells in different population and IL-18. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells. The frequency and ratio of Tregs in TIL of every group on specified time were analyzed and observe the relationship between Tregs and tumor progression by flow cytometry.Results:1 The result of flow cytometry instructed that the mean frequency of Tregs in PBMC of patients with astrocytoma(10.41±2.13%) was higher than that of volunteers(4.35±1.07%, p<0.05). The mean frequency of Tregs in TIL of patients (35.45±2.47%) was higher than that of control(2.53±0.75%, p<0.05). In comparison to autologous patient blood, the frequency of Tregs in tumor tissue were correlated significantly with the malignancy of glioma but age, sex, tumor size and tumor location. There was not a significant difference of the frequency of CD8~+T cells in TILs according to pathological grade.In all patients were divided into higher group(≥M) and lower group( |
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